Revealing ligand-receptor interaction
NMR titration
Jan Novotný
May 3, 2017
Jan Novotný
May 3, 2017 1/17
Introduction of reacting partners
Recommended procedure
O Assignment of free receptor: /3-cyklodextrine (ID H, DQF-COSY) O Assignment of free ligand: doxepine (ID 1H, DQF-COSY, NOESY)
• Identification of proton resonances of ring A and B
• Determination of major and minor conformation of doxepin
O ID NMR titration: rearrangement of /3-cyclodextrin resonaces upon
interaction =>* identification of inner protons O ID NMR titration: rearrangement of doxepin resonaces upon complexation
=>* estimation of binding mode Q ROESY spectrum of complex: ROE intermolecular contacts O Fitting the titration isotherm
ID 1H of /3-cyclodextrin in D 2O
					[4h   oh 1						
					Í (6 \ 5i!i   H2\ u1 hqVJ\x yH 3h    OHo "	7			cd		
	1						e				
	J	f						1 u	b	u	a
			V						u		i
	e c c c T	3 3 3 3					c c u c c	3 * J * 3 * i> e	f 1 t c t c 3 C	n 0 n V	1.0196 J
5.0
4.5
4.0
3.5
3"
00 o
to d
o
OJ
o
o o
[ppm]
Jan Novotný
May 3, 2017
4/17
DQF-COSY of /3-cyclodextrin in D 20
E
Q_ Q.
to
4.5 4.0 82 - 1H (ppm)
Jan Novotný
May 3, 2017       5 / 17
□
-í.oooo;
m	
	
D	
-	
Ill
o
x
-o
INJ
O
[rel]
.....
Ó
p 9
NOESY 700ms of doxepin in D20
NOESY 700ms of doxepin in D20
7.5    /   A \
M   L K J
Jan Novotný
May 3, 2017        10 / 17
ID ľH NMR titration: /3-cyclodextrin
H NMR titration: doxepin
ID *H NMR titration: doxepin
1.9605 1.6154
1.1021
doxepin :(3CD 5:1
3,75:1
1,75:1 1:1
0,75:1
0,2:1 0:1
1.0035
K,L J   | '
1394
0.7815
H-A2
7.4
7.2
7.0
6.8
A
6.6
[ppm]
Find the most perturbed resonances and estimate the time regime of interaction
Jan Novotný
May 3, 2017        14 / 17
ROESY 300ms - doxepin:/3-cyclodextrin=5:l